Aflibercept exhibits VEGF binding stoichiometry di

Authors: Douglas A MacDonald Joel Martin Kathir K Muthusamy JiannKae Luo Erica Pyles Ashique Rafique Tammy Huang Terra Potocky Yang Liu Jingtai Cao Françoise Bono Nathalie Delesque Pierre Savi John Francis Ali Amirkhosravi Todd Meyer Carmelo Romano Meredith Glinka George D Yancopoulos Neil Stahl Stanley J Wiegand Nicholas Papadopoulos

Publish Date: 2016/05/27

Volume: 19, Issue: 3, Pages: 389-406

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Abstract

Antivascular endothelial growth factor VEGF therapies have improved clinical outcomes for patients with cancers and retinal vascular diseases Three antiVEGF agents pegaptanib ranibizumab and aflibercept are approved for ophthalmic indications while bevacizumab is approved to treat colorectal lung and renal cancers but is also used offlabel to treat ocular vascular diseases The efficacy of bevacizumab relative to ranibizumab in treating neovascular agerelated macular degeneration has been assessed in several trials However questions persist regarding its safety as bevacizumab can form large complexes with dimeric VEGF165 resulting in multimerization of the Fc domain and platelet activation Here we compare binding stoichiometry Fcγ receptor affinity platelet activation and binding to epithelial and endothelial cells in vitro for bevacizumab and aflibercept in the absence or presence of VEGF In contrast to bevacizumab aflibercept forms a homogenous 11 complex with each VEGF dimer Unlike multimeric bevacizumabVEGF complexes the monomeric afliberceptVEGF complex does not exhibit increased affinity for lowaffinity Fcγ receptors does not activate platelets nor does it bind to the surface of epithelial or endothelial cells to a greater degree than unbound aflibercept or control Fc The latter finding reflects the fact that aflibercept binds VEGF in a unique manner distinct from antibodies not only blocking the amino acids necessary for VEGFR1/R2 binding but also occluding the heparinbinding site on VEGF165Angiogenesis the growth of new blood vessels from preexisting vasculature is a highly orchestrated process that is critical for proper embryonic and postnatal vascular development 1 Abnormal or pathological angiogenesis is a hallmark of cancer and several retinal diseases where the upregulation of proangiogenic factors such as vascular endothelial growth factor VEGF and placental growth factor PlGF leads to increases in endothelial proliferation changes in vasculature morphology and increased vascular permeability 2 3 In particular blockade of VEGF has shown clinical utility in the oncology setting as well as several retinal vascular diseases characterized by abnormal angiogenesis and/or vascular permeability such as the “wet” form of agerelated macular degeneration AMD the leading cause of blindness in the elderly 4 5 The formation of new blood vessels and vascular leakage in wet AMD leads to macular edema thickening of the retina and loss of vision Elevated levels of VEGF have been found in the vitreous fluid and retinal vasculature of patients with AMD 6 Blocking VEGF activity has also become the therapy of choice for treating diabetic macular edema DME retinal vein occlusions and other ocular diseases where abnormal angiogenesis is the underlying etiology 7 8 9 10Several antiVEGF therapies have been approved for use in neovascular or “wet” AMD Pegaptanib an oligonucleotide aptamer that binds the heparinbinding domain of VEGF165 was the first antiVEGF therapy that showed some benefit in treating wet AMD although most patients still experienced visual decline 11 The introduction of ranibizumab an affinitymatured humanized monoclonal antibody fragment Fab represented a major advance in treating wet AMD patients since it was not only able to stabilize but in many cases improve visual acuity 12 However prior to regulatory approval of ranibizumab ophthalmologists began using bevacizumab a humanized monoclonal antibody to VEGF already approved for metastatic colorectal cancer to treat wet AMD Like ranibizumab bevacizumab binds to all isoforms of VEGFA but is aliquoted and repackaged for offlabel use in ophthalmological indications 13 Several small nonrandomized studies evaluated bevacizumab as a potential treatment option in the treatment for wet AMD and thus began the ongoing debate on the merits of utilizing bevacizumab for the treatment for diseases affecting the ocular vasculature 14 More recently in a large n = 1208 prospective randomized comparison of agerelated macular degeneration treatments trial CATT comparison of the same regimens of ranibizumab or bevacizumab demonstrated similar improvements for the primary end point of visual acuity at 1 year 15 However somewhat higher rates of systemic serious adverse events SAEs were observed in patients treated with bevacizumab compared to ranibizumab Curiously the excess in systemic SAEs observed in bevacizumabtreated patients in the CATT trial did not correspond to adverse cardiovascular events such as hypertension and arteriothrombolic events ATEs previously known to be caused by systemic VEGF inhibition in oncology trials that employed much higher intravenous doses of bevacizumab Rather patients treated intravitreally with lowdose bevacizumab exhibited increases in SAEs affecting other organ systems particularly gastrointestinal disorders While two additional but smaller trials IVAN n = 610 MANTA n = 321 individually showed no statistically significant differences in systemic SAEs between ranibizumab and bevacizumab 16 17 a recent metaanalysis n = 3665 comprising the above studies as well as six additional trials also found a higher incidence of gastrointestinal disorders in patients treated with intravitreal bevacizumab compared to ranibizumab with no differences in other systemic SAEs 18 In comparing the safety profile of ranibizumab to bevacizumab when treating DME the recent Protocol T trial showed no differences in the rates of serious adverse events among all three antiVEGF treatments aflibercept bevacizumab ranibizumab though visual acuity gains with aflibercept were significantly greater than with ranibizumab or bevacizumab in the overall population and these differences were especially evident in patients with poor vision at the start of treatment 19Although mechanisms underlying the potentially higher rate of systemic SAEs not typically associated with antiVEGF activity in patients receiving intravitreal bevacizumab remains unknown a number of investigators have pointed out several fundamental differences between ranibizumab and bevacizumab First ranibizumab is a Fab fragment and is significantly smaller in size than a fulllength antibody This was advantageous for the development for intravitreal injection as the smaller size of ranibizumab was expected to enhance diffusion from the vitreous into the retina and choroid 20 VEGF and closely related molecules are ligands for a family of related receptor tyrosine kinases VEGFRs and as such exist naturally as dimers Since ranibizumab is a Fab fragment two molecules of ranibizumab are bound by each VEGF dimer In contrast bevacizumab is an IgG comprising two Fabs as well as an Fc domain Due to its bivalent nature a single bevacizumab molecule can bind both active sites in a single VEGF dimer Interestingly it has also been reported that bevacizumab has the capacity to form large multimeric complexes with VEGF 21 22 Second though derived from bevacizumab ranibizumab has been affinitymatured with a reported affinity enhancement of 10 to 100fold relative to the Fab which is associated with a similarly enhanced activity of ranibizumab in cellbased assays compared to the Fab fragment of bevacizumab 23 24 However the bivalent nature of the fulllength bevacizumab antibody contributes substantially to its ability to bind and neutralize VEGF Specifically avidity interactions of bevacizumab with its dimeric VEGF target significantly increase binding affinity and potency of the bivalent antibody relative to the monovalent antigenbinding fragment 25 Lastly ranibizumab does not contain a fragment crystallizable Fc region unlike fulllength antibodies While the Fc moiety is useful to increase circulatory halflife when an antibody is given systemically via its interactions with the neonatal receptor the Fc region can also promote effector function if the antibody is multimerized upon binding to its target In the absence of this multimerization the Fc domain of therapeutic antibodies rarely occupies Fcγ receptors due to the constant competition with high levels of endogenous IgG 5–15 g/L 26 Meyer et al 22 have shown that bevacizumab and equimolar amounts of VEGF165 can form immunelike complexes which in the presence of heparin can engage the FcγRIIa receptor on thrombocytes leading to platelet activation Other studies have also reported differences in the activities of ranibizumab and bevacizumab that appear to be unrelated to VEGF neutralization per se For example bevacizumab exhibits enhanced binding to and accumulation in retinal pigment epithelium RPE cells in vitro relative to ranibizumab 27Aflibercept is a novel soluble decoy receptor consisting of an allhuman amino acid sequence comprising the second Ig domain of human VEGFR1 and the third Ig domain of human VEGFR2 expressed as an inline fusion with the constant region Fc of human IgG1 28 Like bevacizumab and ranibizumab aflibercept binds all forms of VEGFA VEGF but in addition binds PlGF and VEGFB 25 Aflibercept was recently approved for the treatment for wet AMD based on the VIEW studies 29 These trials demonstrated equivalent improvements in visual acuity at 1 year between patients treated monthly with ranibizumab and those treated with aflibercept every other month following three initial monthly loading doses Each aflibercept molecule like bevacizumab contains two independent VEGF binding arms held together via the Fc moiety Upon binding VEGF dimers aflibercept could in theory form large multimeric immunelike complexes similar to those previously described for bevacizumab which in turn could trigger processes mediated by lowaffinity Fc receptorsIn the present study we compare the binding stoichiometries of bevacizumab and aflibercept to VEGF165 over a range of relative molar concentrations and also assessed the binding affinities of bevacizumab and aflibercept to VEGF121 VEGF165 and Fcγ receptors alone or when complexed with VEGF The ability of bevacizumabVEGF and afliberceptVEGF complexes to activate human platelets in vitro and the propensity of both bevacizumab and aflibercept to bind to cultured human retinal epithelial ARPE19 and human umbilical vein endothelial cells HUVEC in the presence and absence of VEGF165 and VEGF121 also were assessed The results of these experiments confirm that bevacizumab can form large multimeric complexes with VEGF when both antibody and ligand are present in roughly within tenfold equimolar concentrations In contrast aflibercept forms a homogenous 11 complex with VEGFs over all molar ratios tested Moreover in contrast to the large and heterogenous bevacizumabVEGF165 complexes these discrete afliberceptVEGF165 complexes do not activate platelets in the presence of heparin Finally aflibercept does not bind to the surface of HUVEC or ARPE19 cells to an appreciably greater degree than the control human Fc hFc in the presence or absence of VEGFs In contrast binding of bevacizumab to the surface of these cells was greatly enhanced in the presence of exogenous or endogenous VEGF165 but not VEGF121 This finding suggested that the cell surface binding of bevacizumabVEGF165 complexes was mediated by the heparin and/or neuropilin1 NRP1binding domains of this VEGF isoform This was confirmed by subsequent surface plasmon resonance Biacore experiments which showed that bevacizumabVEGF165 complexes but not afliberceptVEGF165 complexes were readily bound by surfacecaptured NRP1 or heparin In conclusion under certain conditions bevacizumab can form large multimeric immunelike complexes that exhibit enhanced binding not only to a range of Fcγ receptors but also when complexed with VEGF165 to heparin and neuropilin present on the surfaces of cells In contrast aflibercept exclusively forms a 11 complex with VEGF dimers which does not appreciably increase binding to lowaffinity Fc receptors heparin or neuropilin—compared to unbound aflibercept or control monomeric IgGs The fundamental difference between bevacizumab and aflibercept behavior appears to be attributable to the unique way in which the aflibercept encompasses or “Traps” a single VEGF dimer not only blocking the amino acids necessary for VEGFR1/R2 binding but also occluding the heparinbinding site on VEGF as well

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